Recent studies have indicated a role for the Notch family of receptors in mediating cell-fate decisions during hematopoietic development. Studies from our laboratory and others have indicated that these receptors are widely expressed by hematopoietic precursor cells, and that their differentiation is inhibited when they are induced to overexpress the constitutively active cytoplasmic domain of Notch1 or when they are exposed to exogenously presented Notch ligand forms. Preliminary data indicate density-dependent effects of the Notch ligand Deltal on the induction of Notch target gene expression and the determination of hematopoietic stem cell fate. Based on these observations, we hypothesize that 1) the use of different densities of Notch ligands can serve to direct cells toward one of multiple stem cell fate choices by differentially activating Notch target genes, and 2) the unique effects of different Notch ligands in stem cell fate decisions result from differential activation of specific Notch receptors that, in turn, differentially activate Notch target genes. To test these hypotheses, we will use engineered ligands and non-mutant, freshly isolated hematopoietic stem/progenitor populations as a unique approach for rapid activation of Notch signaling, and determine gene expression using microarray analyses and quantitative RT-PCR (Aim 1). The functional importance of target genes associatedwith particular cell fate outcomes will then be determined using knockout mice, gene overexpression and RNA interference (Aim 2). Since effects of ligands depend upon the particular receptor interactions, we will use Notchl and Notch2 conditional knockout mouse models and RNA interference to determine how interactions of Deltal with different Notch receptors lead to different cell fate outcomes (Aim 3). These studies will then be extended to assess the effects of additional Notch ligands, including Jagged 1 and 2, as well as Deltas and 4 (Aim 4). Our long-term goal is to determine the combinations of Notch ligands and receptors, and induced Notch target genes, responsible for governing hematopoietic stem cell fate. Results of such studies promise to have substantial therapeutic implications.